Tetraspanins are small membrane-bound proteins that are expressed in species ranging from sponges to mammals, with each organism expressing a large number of tetraspanin family members. A. Garcia-Espana et al., Appearance of new tetraspanin genes during vertebrate evolution, Genomics (91), 326-334 (2008). Tetraspanins are typically involved in multiple biological processes, such as fertilization, parasite and viral infection, synaptic contacts at neuromuscular junctions, platelet aggregation, maintenance of skin integrity, immune response induction, metastasis suppression, and tumor progression. Tetraspanins cross the membrane four times, but not all four-transmembrane molecules are tetraspanins. In general, tetraspanins have short amino- and carboxy-terminal tails, a small intracellular loop between transmembrane region 2 (TM2) and TM3, a small extracellular loop (EC1) between TM1 and TM2 and a large extracellular loop (EC2) between TM3 and TM4, as illustrated in FIG. 1. See M. Zoller, Tetraspanins: push and pull in suppressing and promoting metastasis, Nature Reviews Cancer (9) 40-55 (2009).
In addition to having these various functionalities within the cell and at the cell membrane, large numbers of tetraspanins are also released from the cell on the surface of exosomes. A. Lakkaraju & E. Rodriguez-Boulan, Itinerant exosomes: emerging roles in cell and tissue polarity, Trends Cell Biol. (18), 199-209 (2008). Exosomes, 30-100 nm vesicles, are released by many cells. J. S. Schorey & S. Bhatnagar, Exosome function: from tumor immunology to pathogen biology, Traffic (9) 871-881 (2008). Exosomes derive from multivesicular bodies, which either fuse with lysosomes or fuse with the plasma membrane and release their intraluminal vesicles as exosomes. The molecular composition of exosomes reflects their origin from intraluminal vesicles and includes several tetraspanins. Id. Exosomal proteins maintain their functional activity, as shown by their capacity to present peptides in major histocompatibility complex class I and II molecules. Id. Exosomes are thought to constitute a potent mode of intercellular communication that is important in the immune response, cell-to-cell spread of infectious agents, and tumour progression. Tetraspanins and their associated proteins are enriched in exosomes. Although the contribution of tetraspanins to the make-up of exosomes is well known, their impact on the functions of exosomes has not been determined. See H. G. Zhang & W. E. Grizzle, A Novel Pathway of Local and Distant Intracellular Communication that Facilitates the Growth and Metastasis of Neoplastic Lesions, American Journal of Pathology (184) 28-41 (2014).
CD9 is a tetraspanin that is broadly expressed in a variety of solid tissues and on a multitude of hematopoietic cells. In particular, CD9 is expressed by endothelial cells, various cells of the central nervous system, vascular smooth muscle cells, cardiac muscle cells, and epithelial cells. M. Zoller, Tetraspanins: push and pull in suppressing and promoting metastasis, Nature Reviews Cancer (9) 40-55 (2009). In addition, CD9 is expressed by platelets (highly expressed), lymphoid progenitor cells, and activated lymphocytes and myeloid cells. Id. and see also H. Maecker et al., The tetraspanin superfamily: molecular facilitators, FASEB Journal (11) 428-442 (1997).
CD9 down-regulation generally correlates with tumor progression in some forms of cancer. However, CD9 is not able to be classified as a metastasis suppressor protein because in some tumor types, the opposite correlation has also been noted, which signals that CD9's relationship with other molecules likely dictates the particular correlations between this molecular and forms of cancer. M. Zoller, Tetraspanins: push and pull in suppressing and promoting metastasis, Nature Reviews Cancer (9) 40-55 (2009). For example, CD9 can hamper metastasis formation by prohibiting integrin-mediated motility. Specifically, in ovarian carcinoma cells, expression levels of CD9 and multiple integrin chains correlate such that the down-regulation of CD9 is associated with weaker matrix adhesion and diffuse growth in vitro. M. Furuya et al., Down-regulation of CD9 in human ovarian carcinoma cell might contribute to peritoneal dissemination, Cancer Research (65) 2617-2625 (2005).
CD9 may also hamper the migration of an isolated, metastasizing cell by multiple modes of action. First, overexpression of CD9 is accompanied by transcriptional downregulation of multiple factors, including WAVE2. WAVE2 is critical in mediating actin polymerization and lamellipoidum and filipodium formation. T. Takenawa & S. Suetsugu, The WASP-WAVE protein network: connecting the membrane to the cytoskeleton, Nature Reviews Molecular and Cellular Biology (8) 37-48 (2007). Also, overexpression of CD9 is known to hamper cell migration through the EGF-EGFR pathway and TGFα. M. Zoller, Tetraspanins: push and pull in suppressing and promoting metastasis, Nature Reviews Cancer (9) 40-55 (2009).
A need exists for anti-CD9 antibodies having unique genetic and amino acid structures, including unique binding and functional characteristics. The development of new anti-CD9 monoclonal antibodies and hybridoma cells lines that produce such monoclonal antibodies would be a valuable tool for the effective diagnosis of various diseases and use in other biomedical techniques.